Near-infrared fluorescent probe for selective detection of glutathione, preparation method and application thereof

Abstract

The present invention discloses a near-infrared fluorescent probe for selectively detecting glutathione, and relates to the field of fluorescent probe technology. The structure of the fluorescent probe is as follows: specifically, 2-fluoro-4-nitrobenzenesulfonate is used as a recognition group, which can specifically recognize glutathione, while other biological thiols (cysteine, homocysteine) and the like have almost no effect. The fluorescent probe of the present invention has the advantage of rapid response, and can achieve rapid and real-time detection of glutathione in cells and living bodies; it has a wide range of applications, and can not only selectively distinguish normal and tumor cancer cells, but also achieve an evaluation of the efficacy of erastin in synergistic effect with DDP at the level of nasopharyngeal cancer cells or living bodies; it is simple to synthesize and prepare, and has low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a near-infrared fluorescent probe for selectively detecting glutathione, a preparation method and an application thereof. Background Art

[0002] Glutathione (GSH) plays important physiological roles in the body, including immune regulation, redox homeostasis, and signal transduction pathways. Generally, GSH levels are higher in tumor cells than in normal cells. Nasopharyngeal carcinoma (NPC) is a malignant tumor that develops on the roof and lateral walls of the nasopharynx. Clinically, it is primarily treated with radiotherapy and chemotherapy. Despite significant side effects, cisplatin (DDP) remains a key chemotherapy option for NPC. DDP binds to specific DNA, affecting cancer cell DNA replication, thereby inhibiting cell proliferation and inducing programmed cell death (apoptosis). Studies have shown that long-term use of this drug can lead to drug resistance in humans. The mechanisms of DDP resistance are related to altered transport and metabolism, enhanced DNA repair or tolerance, and impaired apoptosis. GSH may also be closely linked to DDP resistance. Once DDP is absorbed by cells, GSH binds to DDP, causing it to remain in the cytoplasm, preventing it from entering the nucleus and interacting with target DNA, ultimately leading to DDP resistance. Commonly used chemotherapeutic drugs such as DDP can reduce their toxic effects on cells by binding to intracellular GSH and converting it into excreted metabolites, a process catalyzed by glutathione S-transferase. However, prolonged DDP treatment can also lead to increased intracellular oxidative stress and decreased GSH levels.

[0003] Ferroptosis is a novel form of apoptosis that results in the accumulation of lipid peroxides and a decrease in GSH levels. Studies have shown that the ferroptosis inducer erastin can enhance the sensitivity of tumor cells to chemotherapeutic drugs. Therefore, developing new methods to accurately measure GSH concentration and dynamically monitor changes in GSH levels during the combined treatment of nasopharyngeal carcinoma with erastin and DDP is crucial for a deeper understanding of its underlying mechanisms.

[0004] Many techniques have been used to measure GSH in biological samples, including Raman spectroscopy, high-performance liquid chromatography, and mass spectrometry. Due to the need for complex pretreatment and long analysis time, these analytical methods are not suitable for real-time detection of GSH in living cells and in vivo. Near-infrared fluorescence imaging is a non-invasive biological imaging technology with the following advantages: high sensitivity, high specificity, deeper tissue penetration, versatility, and real-time imaging. This makes the use of near-infrared fluorescent probes to achieve accurate detection of GSH a goal pursued by scientific researchers. So far, people have done a lot of work to develop effective fluorescent probes to track GSH in biological systems, but the following problems still need to be solved: (1) long response time; (2) poor selectivity, for example, 2,4-dinitrosulfonyl ester can selectively recognize biological thiols, but cannot distinguish between cysteine, homocysteine, and glutathione; (3) limited biological application range. So far, there have been no reports on the application of near-infrared GSH fluorescent probes in nasopharyngeal carcinoma cells or in vivo to evaluate the efficacy of erastin synergistically with DDP.

[0005] In view of this, it is very important to develop a technology that can detect GSH concentration with high selectivity, high sensitivity, convenience and rapidity and explore its application in the evaluation of nasopharyngeal carcinoma treatment efficacy. Summary of the Invention

[0006] The present invention provides a near-infrared fluorescent probe for selectively detecting glutathione, as well as a preparation method and application thereof, aiming to solve the problems existing in the above-mentioned background technology.

[0007] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0008] The present invention discloses a near-infrared fluorescent probe for selectively detecting glutathione. The structure of the fluorescent probe is as follows:

[0009]

[0010] The present invention also discloses a method for preparing the aforementioned near-infrared fluorescent probe for selectively detecting glutathione, comprising the following steps:

[0011] (1) 4-diethylamino keto acid was dissolved in methanesulfonic acid, and 1,6-dihydroxynaphthalene was added. The mixture was stirred under heating conditions. After the reaction was completed, the reaction solution was cooled to room temperature, and a dilute hydrochloric acid solution was slowly added dropwise. The mixture was extracted with dichloromethane. The lower organic phase was collected, dried, and spin-dried to obtain the reaction product SNAFL, which was directly used in the next reaction.

[0012] (2) A mixture of SNAFL prepared in step (1), 2-fluoro-4-nitrobenzene-1-sulfonyl chloride and triethylamine was stirred in dry dichloromethane at room temperature. After the reaction was completed, ice water was added to the reaction product, and the organic layer was dried over sodium sulfate, evaporated under reduced pressure, and purified to obtain the desired product SNAFL-GSH.

[0013] In a preferred embodiment of the present invention, in step (1), the molar ratio of the 4-diethylaminoketo acid to the 1,6-dihydroxynaphthalene is 1:2.

[0014] Preferably, in step (1), the reaction temperature is 95° C. and the stirring reaction time is 24 hours.

[0015] Preferably, in step (1), the concentration of the dilute hydrochloric acid is 2 mol / l, and the amount of dilute hydrochloric acid added is 30 ml.

[0016] In another preferred embodiment of the present invention, in step (2), the molar ratio of SNAFL to 2-fluoro-4-nitrobenzene-1-sulfonyl chloride is 1:2.

[0017] Furthermore, in step (2), purification is performed by silica gel column chromatography, and the elution solvent during purification is a methanol-dichloromethane mixture, and the volume ratio of methanol to dichloromethane is 5:95.

[0018] The present invention also discloses the use of the aforementioned near-infrared fluorescent probe in the selective detection of glutathione. Specifically, glutathione can be selectively detected among lysine, glycine, methionine, tryptophan, proline, leucine, serine, arginine, sodium sulfite, sodium sulfate, sodium carbonate, sodium nitrate, potassium chloride, sodium chloride, magnesium sulfate, zinc chloride, hydrogen peroxide, sodium hypochlorite, oxidized glutathione, hydrogen sulfide, homocysteine, cysteine, and glutathione physiologically active substances.

[0019] The present invention also discloses the use of the above-mentioned near-infrared fluorescent probe in detecting endogenous glutathione in nasopharyngeal carcinoma cells. Preferably, the nasopharyngeal carcinoma cells include human nasopharyngeal epithelial cell line NP69, human highly metastatic nasopharyngeal carcinoma cell line 5-8F, human poorly differentiated nasopharyngeal carcinoma cell line CNE-2, and human well-differentiated nasopharyngeal carcinoma cell line CNE-1.

[0020] The present invention also discloses the use of the above-mentioned near-infrared fluorescent probe in evaluating the efficacy of treating nasopharyngeal carcinoma at the cellular level or in vivo level. Preferably, it is used in evaluating the efficacy of erastin in combination with DDP in treating nasopharyngeal carcinoma at the cellular level or in vivo level.

[0021] The specific synthetic route of the present invention is as follows:

[0022]

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The fluorescent probe of the present invention has good selectivity, which solves the problem of poor selectivity of glutathione series fluorescent probes using 2,4-dinitrosulfonyl ester as the recognition group. By using 2-fluoro-4-nitrobenzenesulfonate as the recognition group, it can specifically recognize glutathione, while having almost no effect on other biological thiols (cysteine, homocysteine, etc.);

[0025] 2. The fluorescent probe of the present invention has the advantage of rapid response, which can realize rapid and real-time detection of glutathione in cells and living bodies;

[0026] 3. It has a wide range of applications. It can not only selectively distinguish normal and tumor cancer cells, but also evaluate the efficacy of erastin synergistically with DDP at the nasopharyngeal cancer cell or in vivo level;

[0027] 4. The fluorescent probe is simple to synthesize and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the fluorescence titration diagram of glutathione detected by the fluorescent probe SNAFL-GSH;

[0029] Figure 2 This is the response time diagram of the fluorescent probe SNAFL-GSH to glutathione;

[0030] Figure 3 This is an experimental diagram of the selectivity of the fluorescent probe SNAFL-GSH for glutathione;

[0031] Figure 4 Figure 2 shows the selective discrimination of normal and nasopharyngeal carcinoma cell lines by the fluorescent probe SNAFL-GSH.

[0032] Figure 5 This is a diagram evaluating the efficacy of the fluorescent probe SNAFL-GSH on nasopharyngeal carcinoma at the cellular level;

[0033] Figure 6 This is a diagram evaluating the efficacy of the fluorescent probe SNAFL-GSH on nasopharyngeal carcinoma at the in vivo level. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Any other embodiments obtained by non-creative substitution or transformation of the main design concept of the present invention are within the scope of protection of the present invention.

[0035] Example 1: Synthesis of SNAFL-GSH

[0036]

[0037] 4-Diethylaminoketoacid (939 mg, 3 mmol) was dissolved in methanesulfonic acid (10 ml), and 1,6-dihydroxynaphthalene (960 mg, 6 mmol) was added. The mixture was stirred at 95°C for 24 hours. After the reaction, the reaction solution was cooled to room temperature, and 30 ml of 2 mol / l dilute hydrochloric acid was slowly added dropwise. The mixture was then extracted with dichloromethane, and the lower organic phase was collected, dried, and spin-dried, and used directly in the next reaction. A mixture containing SNAFL (437 mg, 1 mmol), 2-fluoro-4-nitrobenzene-1-sulfonyl chloride (479.2 mg, 2 mmol), and triethylamine (0.5 ml) was stirred in dry dichloromethane (20 ml) at room temperature. After the reaction was completed, ice water was added to the reaction solution. The organic layer was dried over sodium sulfate, evaporated under reduced pressure, and purified by silica gel column chromatography (eluent: 5% methanol / 95% dichloromethane) to give the desired product SNAFL-GSH as a light red solid (390 mg, 61%).

[0038] NMR characterization data: 1 H NMR (400MHz, CDCl3): δ8.59(d,J=9.0Hz,1H),8.16(d,J=8.8Hz,1H),8.11(d,J =8.8Hz,1H),8.05-8.02(m,2H),7.66-7.61(m,2H),7.58(s,1H),7.36(t,J=6.8 Hz,2H),7.13(d,J=7.2Hz,1H),6.81(d,J=8.8Hz,1H),6.62(d,J=8.8Hz,1H),6. 58(s,1H),6.42(d,J=8.7Hz,1H),3.38(q,J=6.7Hz,4H),1.19(t,J=6.8Hz,6H); 13 C NMR (100 MHz, CDCl3): δ 169.54, 160.52, 157.88, 153.32, 152.20, 149.62, 147.87, 147.32, 134.96, 134.61, 132.76, 129.68, 128.92, 126.94, 126.02, 125.27, 125.00, 123.96, 122.89, 122.58, 120.41, 119.51, 119.32, 113.96, 113.48, 113.22, 109.07, 104.65, 97.47, 83.87, 44.48, 12.51; Mass spectral characterization data: HRMS m / z: C 34 H 25FN2O8S[M+H] + calcd for 641.1394found641.1401.

[0039] Example 2: Fluorescence titration of glutathione detected by fluorescent probe SNAFL-GSH.

[0040] like Figure 1 As shown, at room temperature, the fluorescent probe SNAFL-GSH (10 μM) was added to a PBS / DMSO (1:1, volume ratio) solution at pH 7.4. When different concentrations of glutathione were added to the probe solution, the probe fluorescence intensity showed an increasing trend. After the glutathione concentration increased to 1 mM, the fluorescence intensity gradually approached saturation. The fluorescence intensity of the probe SNAFL-GSH at 650 nm showed a good linear response relationship with the glutathione concentration (0.02 mM–0.4 mM) (the linear equation is F 650nm =528.1x+1.646,R 2 =0.99). Using the calculation formula 3σ / k (where k represents the slope of the linear equation and σ represents the standard deviation), the detection limit of the probe for glutathione was calculated to be 1.27 μM. This example demonstrates that the probe SNAFL-GSH can detect glutathione with high sensitivity over a wide concentration range.

[0041] Example 3: Response time of fluorescent probe SNAFL-GSH to glutathione.

[0042] like Figure 2 As shown, the fluorescent probe SNAFL-GSH (10 μM) and glutathione (1 mM) were sequentially added to a PBS / DMSO (1:1, volume ratio) solution (pH 7.4) at room temperature. The fluorescence intensity of the SNAFL-GSH probe at 650 nm was recorded over time, revealing that the fluorescence intensity of SNAFL-GSH reached saturation after 15 minutes. This example demonstrates that the SNAFL-GSH probe can rapidly detect glutathione in vitro.

[0043] Example 4: Selective experiment of fluorescent probe SNAFL-GSH for glutathione.

[0044] like Figure 3As shown, at room temperature, the fluorescent probe SNAFL-GSH (10 μM) was first added to a PBS / DMSO (1:1, volume ratio) solution (pH 7.4). Then, different physiologically active interfering species were added: blank probe, lysine, glycine, methionine, tryptophan, proline, leucine, serine, arginine, sodium sulfite, sodium sulfate, sodium carbonate, sodium nitrate, potassium chloride, sodium chloride, magnesium sulfate, zinc chloride, hydrogen peroxide, sodium hypochlorite, oxidized glutathione, hydrogen sulfide, homocysteine, cysteine, and glutathione. This example demonstrates that the probe SNAFL-GSH is highly selective for glutathione and is capable of selectively distinguishing glutathione from cysteine ​​and homocysteine. In the experiment, only glutathione caused a sharp increase in the probe's fluorescence intensity at 650 nm, while other physiologically active species had little effect.

[0045] Example 5: Selective discrimination of normal and nasopharyngeal carcinoma cell lines using the fluorescent probe SNAFL-GSH.

[0046] like Figure 4 As shown, the ability of the SNAFL-GSH probe to selectively distinguish between normal and cancerous NPC cell lines was validated using the human nasopharyngeal epithelial cell line NP69, the highly metastatic human NPC cell line 5-8F, the poorly differentiated human NPC cell line CNE-2, and the well-differentiated human NPC cell line CNE-1. The SNAFL-GSH probe was incubated with NP69, 5-8F, CNE-2, and CNE-1 at 37°C for 20 minutes, followed by confocal imaging. This example demonstrates the ability of the SNAFL-GSH probe to selectively distinguish between normal and cancerous cells by imaging endogenous glutathione in different cells.

[0047] Example 6: Evaluation of the therapeutic effect of the fluorescent probe SNAFL-GSH on nasopharyngeal carcinoma at the cellular level.

[0048] like Figure 5In this embodiment, human poorly differentiated nasopharyngeal carcinoma cell line CNE-2, human highly differentiated nasopharyngeal carcinoma cell line CNE-1, and human highly metastatic nasopharyngeal carcinoma cell line 5-8F were selected to verify the evaluation of the efficacy of the probe SNAFL-GSH on nasopharyngeal carcinoma at the cellular level. The cell experiment was divided into four groups: 1. Control group, probe SNAFL-GSH (10 μM) was co-incubated with CNE-2, CNE-1, and 5-8F cells at 37°C for 20 minutes; 2. DDP treatment group, CNE-2, CNE-1, and 5-8F cells were first treated with DDP (20 μM) at 37°C for 24 hours, and then the probe SNAFL-GSH (10 μM) was added and incubated for 20 minutes; 3. Erastin treatment group, at 37°C, CNE-2, CNE-1, and 5-8F cells were treated with erastin (5 μM) for 24 hours and then incubated with the probe SNAFL-GSH (10 μM) for 20 minutes. For the DDP / erastin treatment group, CNE-2, CNE-1, and 5-8F cells were first treated with DDP (20 μM) and erastin (5 μM) for 24 hours at 37°C and then incubated with the probe SNAFL-GSH (10 μM) for 20 minutes. The results showed that glutathione levels in the CNE-2, CNE-1, and 5-8F cell treatment groups were lower than those in the control group, and glutathione levels were significantly reduced in the DDP / erastin combination treatment group. This example demonstrates the ability of the probe SNAFL-GSH to assess the therapeutic efficacy of nasopharyngeal carcinoma treatment at the cellular level by monitoring changes in glutathione levels in cancer cells.

[0049] Example 7: Evaluation of the therapeutic effect of the fluorescent probe SNAFL-GSH on nasopharyngeal carcinoma at the in vivo level.

[0050] like Figure 6 As shown, 5-8F cells in the logarithmic growth phase were selected for inoculation into mice. The adherent 5-8F cells were digested with trypsin, and the cell suspension was centrifuged and resuspended in physiological saline. BALB / c nude mice were fixed on the operating table and a volume of 0.2 ml (about 10 cells) was added. 7) were inoculated into the subcutaneous tissue of the armpit. The time of appearance of tumor nodules at the injection site was observed. The long diameter, short diameter and volume of the tumor were measured every week. The experiment complied with animal ethics regulations. After the nasopharyngeal carcinoma mouse model was established, the mice were anesthetized and 50 μl of SNAFL-GSH at a concentration of 50 μM was injected between the tumors for imaging experiments. The mice were randomly divided into three groups: 1. Mice were given erastin at a dose of 10 mg / kg (erastin treatment group); 2. Mice were given cisplatin at a dose of 5 mg / kg (DDP treatment group); 3. Mice were given DDP and erastin at doses of 5 mg / kg and 10 mg / kg (DDP / erastin combined treatment group). All mice were injected intraperitoneally, once every other day, for 2 weeks. After the end of the administration, SNAFL-GSH (50 μM, 50 μl) was injected into the tumor, and the mice were fixed with their limbs facing up for tumor fluorescence imaging without incision. The results showed that tumor-bearing mice showed little fluorescence change after erastin treatment, while mice before and after DDP treatment showed significant fluorescence changes. Compared to mice treated with DDP, mice treated with erastin combined with DDP showed a greater decrease in fluorescence. This demonstrates that erastin may synergistically treat tumors by increasing the sensitivity of cancer cells to DDP. Changes in GSH levels in tumors were positively correlated with the efficacy of DDP-erastin synergistic treatment. The probe SNAFL-GSH has the potential to assess the therapeutic efficacy of nasopharyngeal carcinoma treatment in vivo by monitoring changes in glutathione levels in mouse tumors.

[0051] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0052] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Application of a near-infrared fluorescent probe in the preparation of a reagent for selectively detecting glutathione, wherein the structure of the fluorescent probe is as follows:

2. Application of a near-infrared fluorescent probe in the preparation of a reagent for detecting endogenous glutathione in nasopharyngeal carcinoma cells. The structure of the fluorescent probe is as follows:

3. Application of a near-infrared fluorescent probe in the preparation of a reagent for evaluating the therapeutic efficacy of nasopharyngeal carcinoma at the cellular or in vivo level. The structure of the fluorescent probe is as follows:

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